Chapter 7 5 0 -3 0 Exoribonucleases Jeong Ho Chang, Song Xiang, and Liang Tong Contents 7.1 Introduction .............................................................................. 168 7.2 Sequence Conservation of the XRNs ................................................... 168 7.3 5 0 -3 0 Exonuclease Activity of XRNs .................................................... 170 7.4 Functions of Xrn1 ........................................................................ 172 7.4.1 Functions of Xrn1 in RNA Degradation and Turnover ........................ 172 7.4.2 Functions of Xrn1 in RNA Maturation ......................................... 174 7.4.3 Functions of Xrn1 in DNA Recombination and Chromosome Stability ...... 174 7.4.4 Other Functions of Xrn1 ......................................................... 175 7.5 Functions of Xrn2/Rat1 .................................................................. 175 7.5.1 Functions of Xrn2/Rat1 in RNA Processing and Degradation ................. 176 7.5.2 Functions of Xrn2/Rat1 in RNA Polymerase Transcription Termination ..... 176 7.5.3 Other Functions of Xrn2/Rat1 ................................................... 177 7.6 Protein Partners of XRNs ................................................................ 178 7.7 Functions of XRNs in Plants and Other Organisms .................................... 179 7.8 Overall Structure of Xrn2/Rat1 .......................................................... 179 7.9 Active Site of Xrn2/Rat1 ................................................................ 181 7.10 Structure of the Rat1-Rai1 Complex .................................................... 182 7.11 Rai1/Dom3Z and RNA 5 0 -End Capping ................................................ 183 7.12 The 5 0 -3 0 Exoribonuclease Rrp17 ....................................................... 185 7.13 RNase J1/CPSF-73 ....................................................................... 185 7.14 Perspectives .............................................................................. 186 References ....................................................................................... 186 J.H. Chang • L. Tong (*) Department of Biological Sciences, Columbia University, New York, NY 10027, USA e-mail: [email protected]S. Xiang Department of Biological Sciences, Columbia University, New York, NY 10027, USA Key Laboratory of Nutrition and Metabolism, Institute for Nutritional Sciences, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences, Shanghai 200031, P.R. China A.W. Nicholson (ed.), Ribonucleases, Nucleic Acids and Molecular Biology 26, DOI 10.1007/978-3-642-21078-5_7, # Springer-Verlag Berlin Heidelberg 2011 167
Abstract The 50-30 exoribonucleases have important functions in RNA processing,
RNA degradation, RNA interference, transcription, and other cellular processes.
The Xrn1 and Xrn2/Rat1 family of enzymes are the best characterized 50-30
exoribonucleases, and there has been significant recent progress in the understand-
ing of their structure and function. Especially, the first structural information on
Rat1 just became available. Other 50-30 exoribonucleases have been identified
recently, including yeast Rrp17 and B. subtilis RNase J1, the first enzyme with
50-30 exoribonuclease activity found in prokaryotes. This review will summarize
our current understanding of these enzymes, focusing on their sequence conserva-
tion, molecular structure, biochemical and cellular functions.
7.1 Introduction
Exoribonucleases are involved in RNA processing, RNA degradation, RNA inter-
ference, transcription, modulation of gene expression, antiviral defense, and other
cellular processes. These enzymes can be simply classified based on the direction of
their activity, hence 50-30 or 30-50 exoribonucleases. While a large number of 30-50
exoribonucleases have been identified, in bacteria and eukaryotes (Zuo and
Deutscher 2001) (see also Chap. 8), few 50-30 exoribonucleases are currently
known. The best characterized 50-30 exoribonucleases are the Xrn1/Xrn2 family
of enzymes (to be referred to as XRNs here), which have only been found in
eukaryotes.
Recently, Rrp17 was identified as another 50-30 exoribonuclease, with an impor-
tant role in the 50-end processing of pre-ribosomal RNAs (Oeffinger et al. 2009).
Several enzymes that possess both endo- and 50-30 exoribonuclease activity have
also been reported, including B. subtilis RNase J1 (Condon 2010), the first enzyme
with 50-30 exoribonuclease activity found in prokaryotes (see also Chap. 10). RNaseJ1 is structurally homologous to human CPSF-73 (Mandel et al. 2006), which has
also been suggested to have 50-30 exoribonuclease activity (Dominski et al. 2005) in
addition to its endonuclease activity.
In this chapter, we will focus on the sequence conservation, structure, and
function of the XRNs (Sects. 7.2–7.11). We will also discuss the other 50-30
exoribonucleases, including Rrp17 (Sect. 7.12) and RNase J1/CPSF-73 (Sect. 7.13).
7.2 Sequence Conservation of the XRNs
Yeast and most metazoans have two XRNs, with Xrn1 (175 kD) primarily in the
cytoplasm and Xrn2 (115 kD, more commonly known as Rat1 in yeast) primarily in
the nucleus. RAT1 is an essential gene in yeast, while deletion of XRN1 in yeast
leads to slow growth, sporulation defect, DNA recombination defect, and other
phenotypes. The plant Arabidopsis has three XRNs, two of which (AtXRN2 and
168 J.H. Chang et al.
AtXRN3) are in the nucleus, while the third (AtXRN4) is in the cytoplasm
(Kastenmayer and Green 2000). However, all three Arabidopsis XRNs are Xrn2
homologs, and a sequence homolog of Xrn1 may not exist in higher plants.
The amino acid sequences of the XRNs contain two highly conserved regions
(CR1 and CR2) in their N-terminal segment (Fig. 7.1). The sequence identity
among Xrn2 homologs for these two regions is 50–60%, while that between Xrn1
and Xrn2 homologs is about 40–50%. In comparison, conservation of sequences
outside of these two regions is much lower, especially between Xrn1 and Xrn2. In
fact, the larger size of Xrn1 is due to an extensive C-terminal segment that is absent
in Xrn2. The linker between CR1 and CR2 is also poorly conserved among the
XRNs, both in sequence and in length (Fig. 7.1). Several protease-sensitive sites
identified in S. cerevisiae Xrn1 map to the boundaries of these segments (Fig. 7.1)
(Page et al. 1998).
CR1 covers residues 1–354 of human Xrn1 and residues 1–407 of human Xrn2
(Fig. 7.1), as the latter has three small inserted segments. CR1 contains seven
Asp208, and Asp292 in human Xrn1), and it was recognized that these residues
may be homologous to those in the active site of several other Mg2+-dependent
nucleases (Solinger et al. 1999), even though CR1 shares little overall sequence
conservation with these other enzymes. Therefore, CR1 may have a crucial role in
the active site of the XRNs, which is supported by the fact that mutations of these
acidic residues abolish the exonuclease activity (Johnson 1997; Page et al. 1998;
Solinger et al. 1999). It is expected that the seven conserved acidic residues can
coordinate two Mg2+ ions for catalysis (Yang et al. 2006).
Fig. 7.1 Sequence conservation of XRNs. Schematic drawing of the domain organization of
human Xrn1, S. cerevisiae Xrn1, human Xrn2, S. cerevisiae Rat1, and S. pombe Rat1. The two
highly conserved regions (CR1 and CR2) are labeled. The 570-residue weakly conserved segment
in Xrn1 and a 120-residue segment in Xrn2/Rat1 are indicated. Small triangles in S. cerevisiaeXrn1 indicate protease-sensitive sites. The segment at the extreme C-terminus of these proteins is
not required for activity
7 50-30 Exoribonucleases 169
CR2 covers residues 426–595 of human Xrn1 and residues 509–679 of human
Xrn2 (Fig. 7.1). This segment appears to be unique to the XRNs, and has an
important role in defining the overall landscape of the active site of the XRNs
(see Sect. 7.9).
A 570-residue segment directly following CR2 shows weak sequence conserva-
tion among the Xrn1 enzymes (Fig. 7.1). For example, human and yeast Xrn1 share
26% sequence identity for this segment. In contrast, the remaining C-terminal
segments of the Xrn1 enzymes have little sequence conservation. This C-terminal
segment of yeast Xrn1 is dispensable for its exoribonuclease activity and in vivo
function, while the 570-residue segment, though weakly conserved, is required for
activity (Page et al. 1998).
The Xrn2 enzymes have a roughly 240-residue C-terminal segment following
CR2 (Fig. 7.1). Human Xrn2 and yeast Xrn2/Rat1 share 24% sequence identity for
this segment. The last 125 residues of S. pombe Rat1 can be deleted without
affecting its in vivo function at the permissive temperature (the truncation does
lead to a ts phenotype). Further deletions, removing the C-terminal 204 residues,
inactivated the protein (Shobuike et al. 2001).
Observations on the C-terminal deletion mutants of Xrn1 and Xrn2/Rat1
described above suggest that CR1 and CR2, while highly conserved among the
XRNs, are not sufficient for the activity of these enzymes. A segment following
CR2 (roughly 570 residues for Xrn1 and 120 residues for Xrn2/Rat1) is required for
activity, even though it is only weakly conserved.
The segment of the XRNs containing CR1 and CR2 is generally acidic in nature,
with a pI of 5.6 for this segment of yeast Xrn1. In contrast, the remaining C-terminal
segments of the Xrn1 enzymes are much more basic, with a pI of 9.4 for yeast Xrn1
(Page et al. 1998).
7.3 50-30 Exonuclease Activity of XRNs
The XRNs are Mg2+-dependent, processive 50-30 exoribonucleases (Stevens 1978,1980; Stevens and Poole 1995). Mn2+ can also support the catalytic activity of these
enzymes. They generally prefer single-stranded RNA substrates with a 50-endmonophosphate group. RNAs with a hydroxyl, cap, or triphosphate group at the
50-end are poor substrates for XRNs (Stevens 1978; Stevens and Poole 1995). YeastXrn1 and Rat1 also have weak exonuclease activity toward single-stranded DNA
(Page et al. 1998; Solinger et al. 1999; Stevens and Poole 1995). Yeast Xrn1 can
cleave G4 tetraplex DNA derived from guanine-rich sequences that are normally
found in telomeres (Liu and Gilbert 1994), while mouse Xrn1 can also cleave G4
tetraplex RNA (Bashkirov et al. 1997).
The presence of strong secondary structures in the RNA substrate can block or
stall the exoribonuclease activity of yeast Xrn1 and Rat1 (Poole and Stevens 1997;
Stevens and Poole 1995). A strong stem loop at the 50-end of the genome of
170 J.H. Chang et al.
Narnavirus 20 S RNA, a persistent virus in yeast, protects it from degradation by
Xrn1 (Esteban et al. 2008).
The exoribonuclease activity of yeast Xrn1 and Rat1 is inhibited by adenosine
30,50 bisphosphate (pAp) (Dichtl et al. 1997). Nearly 80% inhibition of both Xrn1
and Rat1 can be achieved with 1 mM pAp. The inhibition of Xrn1 is not affected by
the concentration of the RNA substrate, suggesting that pAp may not compete
against RNA. pCp and pUp are as potent as pAp in inhibiting Xrn1, while 50 or 30
AMP is essentially inactive. pAp is a byproduct of the sulfate assimilation pathway,
and is normally converted to 50 AMP and Pi by the enzyme 30,50 bisphosphatenucleotidase, Hal2/Met22 in yeast. Hal2 is inhibited by submillimolar
concentrations of Li+, and the resulting increase in cellular pAp concentration (up
to 3 mM) and the consequent inhibition of Xrn1 and Rat1 may be part of the
mechanism for Li+ toxicity in yeast. A similar mechanism may contribute to the
physiological effects of Li+ in other organisms, including the therapeutic effects of
Li+ for the treatment of various neurological diseases in humans.
The cellular functions of the XRNs are primarily linked to their exoribonuclease
activity. Therefore, these enzymes are involved in the turnover of mRNAs and
degradation of aberrant mRNAs (quality control) (Fig. 7.2). They are also involved
in the maturation (50 trimming) of ribosomal RNAs (rRNAs), small nucleolar RNAs
(snoRNAs), and others, as well as the degradation of hypomodified mature tRNAs
and spacer RNA byproducts from rRNA processing. The exoribonuclease activity
of Xrn2/Rat1 also contributes to transcription termination by nuclear RNA
polymerases I and II (Pol I and Pol II). The physiological functions of Xrn1 and
Cytoplasm
Nucleus
P bodyNucleolus mRNA
cap
5′
5′
5′3′
5′
5′
5′
5′
5′
5′
5.85
255rRNA Poly (A)
Rail1 hypomodifiedtRNA
cap Dcp1/2
Lsm1-7Dhh-1
Pat1
PTC
PTC
N N
N N
RISC
mRNA(siRNA)
Ago
Exosome
Rtt 103
CTD
Telomerase
TERRARNAPol.
Xrn1
Rat1/Xrn2
Fig. 7.2 Schematic drawing of the functions of XRNs
7 50-30 Exoribonucleases 171
Xrn2/Rat1 will be described in more detail in the following two sections, and the
functions of the plant XRNs are decribed in Sect. 7.7.
Some of the functional differences between Xrn1 and Xrn2/Rat1 are due to their
different cellular localizations. However, a nuclear-targeted Xrn1 can rescue the
lethal phenotype of rat1-1 (carrying a ts mutation in RAT1) yeast cells, suggestingthat Xrn1 can complement the essential function of Rat1 (Johnson 1997). Con-
versely, RAT1 expressed from a high copy-number plasmid, as well as Rat1 without
its nuclear localization sequence (NLS), can rescue the defects due to the loss of
Xrn1 (Johnson 1997).
The XRNs may have other functions that are independent of their exonuclease
activity. For example, they may mediate protein–protein interactions to recruit
other proteins or to be recruited by other proteins and/or RNA to proper locations
in the cell. Especially, yeast Rat1 is known to form a stable complex with Rai1
(Rat1 interacting protein 1), which can stimulate the exoribonuclease activity of
Rat1. Rat1 may also interact with other protein factors that are important for Pol II
termination, including Rtt103. Yeast Xrn1 may interact directly with microtubules.
The protein complexes for Xrn1 and Xrn2/Rat1 are described in a Sect. 7.6.
7.4 Functions of Xrn1
Xrn1 nuclease activity was first identified in yeast (Larimer et al. 1992; Stevens
1978). Later studies showed that the enzyme is identical to several other proteins
isolated based on other biochemical and functional properties (Kearsey and Kipling
1991), DNA strand exchange protein 1 including (Sep1) (Tishkoff et al. 1991),
DNA strand transferase 2 (Dst2) (Dykstra et al. 1991), Kar� enhancing mutant 1
(Kem1) (Kim et al. 1990), and radiation-resistant on 5 (Rar5) (Kipling et al. 1991).
Xrn1 may also be identical to the antiviral superkiller 1 (Ski1) protein (Johnson and
Kolodner 1995). This illustrates the various functions for this enzyme other than
RNA metabolism, such as DNA recombination, chromosome stability, microtubule
association, nuclear fusion, meiosis, telomere maintenance, and cellular senes-
cence. Defects in many of these processes are observed in cells lacking Xrn1
(Larimer and Stevens 1990).
Xrn1 homologs in S. pombe (also known as Exo II) (Kaslin and Heyer 1994) andhigher eukaryotes have also been cloned, including C. elegans (Newbury and
Woollard 2004), Drosophila (Pacman) (Till et al. 1998), mouse (Bashkirov et al.
1997), and humans (Sato et al. 1998; Shimoyama et al. 2003).
7.4.1 Functions of Xrn1 in RNA Degradation and Turnover
Xrn1 has important roles in mRNA degradation and turnover. This subject has been
reviewed extensively over the past few years (Conti and Izaurralde 2005; Doma and
172 J.H. Chang et al.
Parker 2007; Houseley and Tollervey 2009; Isken and Maquat 2007; Parker and
Song 2004), and will only be discussed briefly here, focusing on the functions of
Xrn1 in these processes. The basic mode of action is that RNAs with a 50-endmonophosphate are generated by decapping of mRNAs (possibly preceded by
deadenylation) or by endonucleolytic cleavage, which are then rapidly degraded
by Xrn1 (Fig. 7.3). The 30-50 exosome also plays an important role in mRNA
metabolism (see Chap. 9), although Xrn1 is the primary enzyme for mRNA
degradation and turnover in yeast. The rate of mRNA turnover is reduced in yeast
cells lacking Xrn1, leading to accumulation of non-polyadenylated mRNAs that
also partially lack the 50-end cap structure (Hsu and Stevens 1993).
Xrn1 is predominantly localized to cytoplasmic foci known as P-bodies
(processing bodies/GW bodies), which are the major location for mRNA decapping
and 50-30 degradation as well as for temporary storage of mRNAs during translation
repression (Kulkarni et al. 2010; Parker and Sheth 2007). Recent studies show that
decapping and Xrn1-mediated degradation of mRNAs can also occur on actively
translating ribosomes (Hu et al. 2009), as does deadenylation-independent
decapping initiated by nonsense-mediated decay (NMD) (Hu et al. 2010).
Endonucleolytic cleavage of mRNAs can be initiated by no-go decay (NGD) and
by the RNA-induced silencing complex (RISC) for RNA interference (RNAi)
(Fig. 7.3) (Orban and Izaurralde 2005). In addition, endonucleolytic cleavage
during maturational processing of many RNA precursors can produce byproducts
that are degraded by Xrn1. For example, Xrn1 degrades the internal transcribed
spacer ITS1 generated from pre-ribosomal RNA processing in yeast (Fig. 7.4)
(Stevens et al. 1991).
5′ cap ORF
Dcp1/2
Dcp1/2
Xrn1
Xrn1
Xrn1
Exosome
Exosome
Deadenylasecompex
Deadenylation dependent(mRNA turnover)
Deadenylation independent(NMD)
Endonuclease dependent(NGO, NMD, IRE1/PMR1, RNAi)
Poly (A)
Fig. 7.3 Schematic drawing of mRNA turnover and mRNA degradation pathways
7 50-30 Exoribonucleases 173
Recently, it has been found that Xrn1 and Rat1 can degrade hypomodified
mature tRNAs in yeast, in the rapid tRNA decay (RTD) pathway (Chernyakov
et al. 2008).
7.4.2 Functions of Xrn1 in RNA Maturation
Xrn1 plays a role in pre-ribosomal RNA processing and maturation, which may be
especially important in the absence of Rat1 activity in yeast. This will be discussed
in more detail in Sect. 7.5.1.
7.4.3 Functions of Xrn1 in DNA Recombinationand Chromosome Stability
Xrn1 was identified in a biochemical search for DNA recombination proteins (and
hence named Sep1 and Dst2) (Dykstra et al. 1991; Tishkoff et al. 1991). It has
homologous pairing and strand exchange activities in vitro. Yeast cells lacking
Xrn1 are defective for intrachromosomal recombination, sporulation, and trigger
Fig. 7.4 Schematic drawing of the pre-ribosomal RNA processing pathways. The extent of the
exonuclease trimming is indicated by the arrows
174 J.H. Chang et al.
arrest at pachytene stage in the meiotic cell cycle (Solinger et al. 1999; Tishkoff
et al. 1995). On the other hand, Xrn1 may not be involved in mitotic recombination
or mating-type switching.
Xrn1 was identified from a genetic screen for mutants that can enhance the
nuclear fusion defect of yeast cells carrying the kar1-1 mutation (hence named
Kem1) (Kim et al. 1990). Kem1 mutants also have reduced chromosome stability
and are hypersensitive to the microtubule-destabilizing drug benomyl. Defective
interactions with microtubules may be the basis of these phenotypes (see Sect. 7.6).
Yeast cells lacking Xrn1 also show cellular senescence and telomere shortening
(Liu et al. 1995), which may be related to the nuclease activity of this enzyme
toward G4 tetraplex DNA (Liu and Gilbert 1994).
Most of the defects in these nuclear processes (sporulation defect, arrest at
pachytene, chromosome instability) due to loss of Xrn1 can be rescued by targeting
Rat1 to the cytoplasm (Johnson 1997); consistent with the fact that Xrn1 is
primarily a cytoplasmic protein. This also suggests the possibility that the effects
of Xrn1 on these processes may not be direct.
7.4.4 Other Functions of Xrn1
Human Xrn1 may function as a tumor suppressor in osteogenic sarcoma, and its
expression level is reduced in these tumors (Zhang et al. 2002). Mouse Xrn1 is
highly expressed in testis, suggesting a functional role in gametogenesis (Shobuike
et al. 1997). Drosophila Xrn1/Pacman is required for male fertility (Zabolotskaya
et al. 2008). The expression level of Pacman is correlated with developmental
stages in Drosophila (Till et al. 1998), and C. elegans Xrn1 is critical for ventral
epithelial enclosure during embryogenesis (Newbury and Woollard 2004).
Xrn1 is also involved in host antiviral response. It can suppress viral RNA
recombination (Cheng et al. 2006), and down-regulate replication by HIV
(Chable-Bessia et al. 2009) and HCV (Jones et al. 2010).
7.5 Functions of Xrn2/Rat1
Like Xrn1, Xrn2 was first identified from several independent studies, due to its
different functions. It was found from a screen for ribonucleic acid trafficking
defects in yeast, and hence named Rat1 (Amberg et al. 1992), and from a screen
for protein translation defects (Hke1, homology to Kem1), which are more likely
due to defects in RNA processing or trafficking (Kenna et al. 1993). It was also
found to have functions in transcription activation (Tap1) (Aldrich et al. 1993; di
Segni et al. 1993).
In contrast to XRN1, RAT1 is an essential gene in yeast, although the exact
function (or the substrate) of this protein that is required for cell viability is
currently not known.
7 50-30 Exoribonucleases 175
Homologs of Rat1/Xrn2 in other organisms have also been cloned, including
S. pombe (also named Dhp1) (Shobuike et al. 2001; Sugano et al. 1994), mouse
(Dhm1) (Shobuike et al. 1995), and humans (Zhang et al. 1999).
7.5.1 Functions of Xrn2/Rat1 in RNA Processingand Degradation
Rat1 is required for 50-end trimming during the maturation of the 5.8 S and 25 S
rRNA, and Xrn1 can support this activity in the absence of Rat1 (El Hage et al.
2008; Fang et al. 2005; Fatica and Tollervey 2002; Geerlings et al. 2000; Henry
et al. 1994). The 5.8 S, 18 S and 25 S ribosomal RNAs are made in a single
transcript by Pol I in eukaryotes, which undergoes extensive endo and
exonucleolytic processing (Fig. 7.4). The primary transcript includes two external
transcribed spacers (50- and 30-ETS) and two internal transcribed spacers (ITS1 andITS2) (Fig. 7.4). Rat1/Xrn1 is involved in the degradation of a fragment of ITS1
that is released during pre-rRNA processing. Recent studies identified Rrp17 as an
independent 50-30 exoribonuclease that can also process the 50-ends of 5.8 S and
25 S rRNA (see Sect. 7.12) (Oeffinger et al. 2009).
Rat1 is required for 50-end processing of polycistronic and some intronic
snoRNAs in yeast, and Xrn1 can (at least partially) support this activity (Lee
et al. 2003; Petfalski et al. 1998; Qu et al. 1999; Villa et al. 1998). Rat1 and Xrn1
are involved in the degradation of some intron-containing unspliced pre-mRNAs
and intron lariats (Danin-Kreiselman et al. 2003). The entry sites for the XRNs are
produced by prior endonucleolytic cleavage or by debranching of the intron lariat.
Rat1 degrades telomeric repeat-containing RNA (TERRA) in yeast (Luke et al.
2008). Telomeres are transcribed by Pol II and polyadenylated, and cells lacking
Rat1 accumulate TERRA and have short telomeres. Therefore, Rat1 promotes
telomere elongation and is important for telomerase regulation.
7.5.2 Functions of Xrn2/Rat1 in RNA Polymerase TranscriptionTermination
Xrn2/Rat1 has a central role in the torpedo model for transcription termination by
RNA polymerases I and II. This area has been reviewed extensively over the past
few years (Buratowski 2005; Ghazal et al. 2009; Gilmour and Fan 2008; Luo and
Bentley 2004; Richard and Manley 2009; Rondon et al. 2009), and will only be
briefly discussed here.
The torpedo model suggests that the downstream RNA product, with a
50-monophosphate, produced by an endonucleolytic cleavage of the primary tran-
script serves as the entry point for a 50-30 exoribonuclease, which degrades
this downstream RNA, catches up to the elongating (or paused) polymerase, and
176 J.H. Chang et al.
causes transcription termination (Connelly and Manley 1988). The 50-30
exoribonuclease for Pol II termination was identified as Rat1 in yeast and Xrn2 in
mammalian cells (Kim et al. 2004; West et al. 2004). It was shown more recently
that Pol I transcription termination is also mediated by the torpedo model, with Rat1
being the 50-30 exoribonuclease for this function in yeast (Fig. 7.5) (El Hage et al.
2008; Kawauchi et al. 2008).
The molecular mechanism for how Xrn2/Rat1 brings about transcription termi-
nation once it catches up to the polymerase is still not clearly understood. Degrada-
tion of the downstream product is not sufficient for termination. Nuclear-targeted
Xrn1 can degrade the downstream product in yeast cells lacking Rat1, but nuclear
Xrn1 cannot cause Pol II termination (Luo et al. 2006). In addition, Rat1 alone is not
sufficient for Pol II termination in an in vitro transcription system (Dengl and
Cramer 2009). Therefore, other factors are also required for transcription termina-
tion by Rat1/Xrn2. The pre-mRNA 30-end processing factor Pcf11 may be impor-
tant for dismantling the polymerase elongation complex (Luo et al. 2006; West and
Proudfoot 2008; Zhang et al. 2005).
7.5.3 Other Functions of Xrn2/Rat1
Xrn2 is a candidate gene for spontaneous lung tumor susceptibility based
on a genome-wide association study in mice (Lu et al. 2010). In addition,
polymorphisms in human Xrn2 are associated with human lung cancer, and
over-expression of human Xrn2 can affect the differentiation of a leukemia cell
line (Park et al. 2007).
Fig. 7.5 Schematic drawing of the allosteric-torpedo (unified) model of Pol II termination.
Changes in the phosphorylation state of the CTD and in the body of Pol II are indicated (Modified
from Luo et al. 2006)
7 50-30 Exoribonucleases 177
7.6 Protein Partners of XRNs
Xrn1 is associated with the decapping machinery in yeast and may directly interact
with several of its components, including Dcp1/Dcp2, Pat1, Dhh1, and the Lsm1–7
complex (Coller and Parker 2004). This may facilitate the degradation of RNAs
once they are decapped by this machinery. The region(s) of Xrn1 that is required for
these interactions has not been identified.
Yeast Xrn1 interacts directly with tubulin and promotes microtubule assembly
(Interthal et al. 1995). Cells lacking Xrn1 show increased chromosome loss, defects
in spindle pole body separation and karyogamy, and hypersensitivity to benomyl
(Kim et al. 1990). The exonuclease activity of Xrn1 is not required for this
interaction (Solinger et al. 1999). The benomyl sensitivity of cells lacking Xrn1
can be rescued by targeting Rat1 to the cytoplasm, although cytoplasmic Rat1 does
not appear to be associated with microtubules (Johnson 1997).
In yeast, Rat1 has direct and strong association with Rai1, and the Rat1-Rai1
complex was first purified from S. cerevisiae extract (Stevens and Poole 1995).
A stable Rat1-Rai1 complex was also observed in S. pombe (Shobuike et al. 2001).Rai1 (45 kD) has orthologs in most eukaryotes, including plants, and the mamma-
lian homolog is known as Dom3Z (Xue et al. 2000). The sequences of these
orthologs are highly divergent, however, with only a few conserved residues. In
contrast to Rai1, Dom3Z does not appear to interact with Xrn2.
Rai1 is not essential for yeast cell viability, and does not have any nuclease
activity (Xue et al. 2000). However, Rai1 can moderately stimulate the
exoribonuclease activity of Rat1 (Xiang et al. 2009; Xue et al. 2000). This may
be due in part to the stabilization of Rat1 by Rai1. Rat1 is unstable and quickly loses
activity upon pre-incubation at 30 �C, whereas the Rat1-Rai1 complex is able to
retain most of its nuclease activity during this pre-incubation (Xue et al. 2000). Like
Rat1, Rai1 is also required for 5.8 S rRNA processing. However, while Rat1 is only
involved in the 50-end processing of this RNA, Rai1 is also needed for 30-endprocessing (Fang et al. 2005; Xue et al. 2000).
The Drosophila genome contains two homologs of Rai1/Dom3Z: CG9125 and
CG13190. CG13190, also known as Cutoff (Cuff), was first identified in a female-
sterile screen. cuffmutations affect germline cyst development, produce ventralized
eggs, and reduce female fecundity (Chen et al. 2007). Over-expressed Cuff is
localized in the cytoplasm and in perinuclear puncta, and Cuff does not interact
with Drosophila Xrn2.
S. cerevisiae also has a homolog of Rai1, Ydr370c, which is poorly conserved
with Rai1 at the sequence level (Xue et al. 2000). The function of this protein is
currently not known. Sequence analysis suggests that this homolog is restricted to
only a few of the fungal species, while most other eukaryotes contain only one
homolog of Rai1/Dom3Z.
Rtt103 (regulation of Ty1transposition 103) can interact with the Rat1-Rai1
complex in yeast (Dengl and Cramer 2009; Kim et al. 2004). Rtt103 was originally
found by a screen for mutants that increased Ty1 transposon’s mobility (Scholes
178 J.H. Chang et al.
et al. 2001). Rtt103 has a RNA Pol II carboxy-terminal domain (CTD)-interacting
domain (CID), and recognizes Ser2 phosphorylated CTD. Rtt103 may be involved
in nuclear pre-mRNA regulation (Kim et al. 2004), and it localizes at the 30-end of
transcribing genes together with Rat1-Rai1 in vivo (Kim et al. 2004) and in vitro
(Dengl and Cramer 2009).
A functional interaction between Rat1 and the pre-mRNA 30-end processing
factor Pcf11 has been suggested (Luo et al. 2006; West and Proudfoot 2008),
although currently there is no biochemical evidence for direct interaction between
these two proteins. Pcf11 may be responsible for the recruitment of Rat1 to the 30-end of pre-mRNAs and/or vice versa.
7.7 Functions of XRNs in Plants and Other Organisms
In Arabidopsis, AtXRN2 is involved in 50-end processing of 5.8 S and 25 S rRNAs
(Zakrzewska-Placzek et al. 2010), a function similar to that of Rat1. In addition,
both AtXRN2 and AtXRN3 can degrade miRNA loop and transgene for
suppressing endogenous post-transcriptional gene silencing (Gy et al. 2007).
The cytoplasmic AtXRN4 can degrade specific RNA transcripts but may not be a
general RNA degradation enzyme, in contrast to Xrn1. It degrades 30-end mRNA
products derived from miRNA-mediated cleavage (Souret et al. 2004). Mutation of
AtXRN4 leads to accumulation of decapped mRNAs that could be templates for
facilitating the RNAi pathway, and AtXRN4 may link mRNA degradation and
RNA silencing (Gazzani et al. 2004; Gregory et al. 2008). AtXRN4 also contributes
to the regulation of the ethylene response pathway (and hence is also known as
EIN5, ETHYLENE-INSENSITIVE5) (Olmedo et al. 2006; Potuschak et al. 2006).
In Trypanosoma brucei and other kinetoplastids, four XRN-related proteins havebeen identified, XRNA, XRNB, XRNC, and XRND (Li et al. 2006). XRND is
nuclear, XRNB and XRNC are cytoplasmic, and XRNA is present in both
compartments. XRNA and XRND are essential for growth, and XRNA is required
for degrading highly unstable, developmentally regulated mRNAs, while having
little effect on more stable, unregulated mRNAs (Li et al. 2006).
7.8 Overall Structure of Xrn2/Rat1
Crystal structure of the S. pombe Rat1-Rai1 complex is the first structural informa-
tion on the XRNs (Xiang et al. 2009). The structure of Rat1 indicates that CR1 and
CR2 form a single, large domain (Fig. 7.6a). CR1 is composed of a seven-stranded
(b1 through b7) mostly parallel b-sheet sandwiched by a-helices on both faces.
Strands b2 through b7 are arranged similar to those in the Rossmann fold, but with
strand b7 running in the opposite direction. A helix is inserted after b2 (aΒ) and b7(aD). CR2 contains several helices and long loops, which wrap around the base
7 50-30 Exoribonucleases 179
(N-terminal end) of the aD helix. Residues in the linker between CR1 and CR2 are
mostly disordered in the structure. The N- and C-termini of this segment are located
within 10 A of each other, suggesting that it is likely an inserted cassette between
the two conserved regions (Fig. 7.6a).
A striking feature of the S. pombe Rat1 structure is the long aD helix, with its
C-terminus projected 30-A away from the rest of the structure (Fig. 7.6a). This
feature has been named the “tower domain.” The N-terminal residues of helix aDare strongly conserved among XRNs, and they contribute to the formation of
the active site (see Sect. 7.9). The C-terminal residues of this helix are poorly
Fig. 7.6 Structure of the S. pombe Rat1–Rai1 complex. (a) Schematic drawing of the structure of
S. pombe Rat1–Rai1 complex (Xiang et al. 2009). The active site of Rat1 is indicated with the star,
and the arrow points to the opening of the Rai1 active site pocket. A bound divalent metal cation in
the active site of Rai1 is shown as a sphere. (b) Schematic drawing of the active site of S. pombeRat1. Side chains of residues in the active site are shown and labeled. Overall molecular surface of
(c) Rat1, (d) FEN-1 (Chapados et al. 2004), and (e) T4 RNase H (Devos et al. 2007). The active
site is indicated with the star
180 J.H. Chang et al.
conserved, and sequence analysis indicates that this helix is much shorter in Xrn1.
Two temperature-sensitive mutations in XRNs, P90L in Xrn1 (Page et al. 1998),
and Y657C in Rat1 (the rat1-1 mutation) (Luo et al. 2006), are located near the
N-terminal end of helix aD. Both mutations may destabilize this helix at the non-
permissive temperature, supporting the functional importance of the tower domain.
The structure of CR1 has many homologs, most of which are nucleases. These
include the FEN-1 family of endonucleases (Chapados et al. 2004; Hwang et al.
1998; Sakurai et al. 2005; Sayers and Artymiuk 1998), the 50 exonuclease from the
phage T5 (Ceska et al. 1996), RNase H from the phage T4 (Devos et al. 2007;
Mueser et al. 1996), the 50 nuclease domain of Taq DNA polymerase (Kim et al.
1995; Murali et al. 1998), and other PIN domain-containing nucleases (Clissold and
Ponting 2000; Glavan et al. 2006). The sequence conservation between Rat1 and
these other enzymes is very low, between 8% and 15%. The structural homology is
limited to strands b2-b7 in the central b-sheet and a few of the flanking helices. The
tower domain in Rat1 is equivalent to the helical clamp in A. fulgidus FEN-1
(Chapados et al. 2004) and the helical arch in T5 exonuclease (Ceska et al. 1996),
but the equivalent region is a long loop inM. jannaschii FEN-1 (Hwang et al. 1998)and is disordered in T4 RNase H (Devos et al. 2007; Mueser et al. 1996).
The Rat1 structure covers residues 1–874, which are sufficient for the activity of
this protein at the permissive temperature (Shobuike et al. 2001). The 120-residue
segment following CR2 forms three distinct structural features (Fig. 7.6a). The
N-terminal region (residues 752–840) of this segment adds four anti-parallel strands
(b8–b11) to the central b-sheet of CR1, producing a highly twisted 11-stranded
b-sheet. Residues 841–863 form a long loop that traverses the entire bottom face of
the central b-sheet of CR1. Finally, the C-terminal region of this segment (residues
864–874) forms an a-helix that interacts with helices aA and aH in CR1. Therefore,
despite being poorly conserved among XRNs, this segment has an important struc-
tural role, which may explain why it is required for the function of Rat1.
The strong sequence conservation for CR1 and CR2 suggests that these two
segments should have a similar structure in Xrn1 (with the exception of the tower
domain). On the other hand, most of the 570-residue segment following CR2 is
unique to Xrn1 and forms several distinct structural domains, as revealed by the
structure of Xrn1 (unpublished data).
7.9 Active Site of Xrn2/Rat1
The active site of Rat1 is located at the top of the central b-sheet of CR1, withcontributions from residues at the base of the aD helix (Fig. 7.6a). The seven
conserved acidic residues in CR1 form a cluster, and are located in the center of
the active site (Fig. 7.6b). In the structure of bacteriophage T4 RNase H, two metal
ions are associated with these acidic residues (Mueser et al. 1996), consistent with
the hypothesis that the two metal ions mediate the nuclease activity (Yang et al.
2006). Metal ions were not observed in the structure of Rat1, and there are some
7 50-30 Exoribonucleases 181
noticeable differences in the conformations of some of these acidic side chains
between Rat1 and T4 RNase H.
Three positively-charged (Lys111, Arg118, Arg119) and two polar (Gln114,
Gln115) residues at the base of the aD helix, as well as His61, His65, and Asn57 in
helix aB contribute their side chains to the active site (Fig. 7.6b). These residues
form a steep wall at one side of the active site, and may be important for interacting
with the phosphate backbone of the RNA substrate. Mutations of these residues, as
well as several of the conserved acidic residues, disrupt Rat1’s exonuclease
activity.
Residues in CR2 encircle the base of helix aD, but contribute few residues to the
Rat1 active site. The side-chain hydroxyl groups of Tyr627 and Tyr628 hydrogen-
bond with the acidic residues Glu205 and Asp237 in the active site, respectively,
and the side chain of Gln671 is located in the cluster of polar side chains from the
aB and aD helices (Fig. 7.6b).
However, CR2 introduces a dramatic difference in the overall landscape of the
active site of Rat1 as compared to other related nucleases. Due to the presence of
CR2, the Rat1 active site is a pocket (Fig. 7.6c), while the active sites of related
nucleases are more open (Figs. 7.6d,e). It has been suggested that the ssDNA
substrate threads through the helical arch in T5 nuclease (Ceska et al. 1996). In
T4 RNase H, a single-stranded DNA portion of its forked DNA substrate is also
bound in this region (Devos et al. 2007). However, such a binding mode would not
be possible in Rat1, as the substrate would clash with residues in CR2. This may be
the explanation why Rat1 is an exonuclease.
The poorly conserved C-terminal segment of Rat1, following CR2, is located
away from the active site and does not have any direct contributions to it. However,
this segment is important for recruiting Rai1, which can (indirectly) stimulate the
exoribonuclease activity of Rat1.
7.10 Structure of the Rat1-Rai1 Complex
The structure of the Rat1-Rai1 complex shows that Rai1 is bound on the opposite
face from the Rat1 active site (Fig. 7.6a), interacting primarily with the poorly
conserved C-terminal loop that traverses the bottom of CR1 (Xiang et al. 2009).
The Rat1-Rai1 interface buries approximately 800 A2 of surface area of each
protein, consistent with the stability of this complex. Ion-pair, hydrogen-bonding,
as well as van der Waals interactions contribute to the formation of this complex.
Mutations introduced in this interface can abolish the interaction as well as the
stimulation of Rat1 by Rai1 (Xiang et al. 2009).
Rai1 does not directly contribute to the active site of Rat1. Structural and
biochemical studies indicate that Rai1 enhances Rat10s exonuclease activity at
least in part by increasing the enzyme’s stability (Xue et al. 2000). This is also
supported by the observation that over-expressing Rai1 can rescue a temperature-
sensitive phenotype of Rat1 (Shobuike et al. 2001). On the other hand, real-time
182 J.H. Chang et al.
measurements of exoribonuclease activities of Rat1 and Rat1-Rai1 complex sug-
gest that the Rat1 enzyme is inherently less active (Sinturel et al. 2009). Therefore,
Rai1 may also indirectly help to organize the active site of Rat1. The structure of
Rat1 alone, and comparison with the Rat1–Rai1 complex, may reveal any changes
in the active site that is induced by Rai1 binding.
Residues at the Rat1–Rai1 interface are generally conserved among the fungal
proteins, consistent with the observations that Rat1 and Rai1 form tight complexes
in both S. cerevisiae and S. pombe (Shobuike et al. 2001; Stevens and Poole 1995).However, Rai1 residues that interact with Rat1 are not conserved in the mammalian
Rai1 homolog Dom3Z, and Dom3Z does not interact with mammalian Xrn2.
Therefore, the Rat1–Rai1 interaction appears to be unique to the fungal species.
Whether mammalian Xrn2 also has a protein partner that can stimulate its activity is
currently not known.
7.11 Rai1/Dom3Z and RNA 50-End Capping
An unexpected discovery from the structure of Rai1 is that it contains a large pocket
(Figs. 7.7a,b), and the few residues that are highly conserved among Rai1 orthologs
are all located in this pocket (rather than in the interface with Rat1) (Xiang et al.
2009). Moreover, three conserved acidic side chains, Glu150, Asp201, and Glu239
(S. pombe Rai1 numbering), together with the main-chain carbonyl of Leu240 and
two water molecules octahedrally coordinate a divalent cation (Mg2+ or Mn2+)
(Fig. 7.7c), and this metal ion is located near the bottom of the pocket (Fig. 7.7b).
Therefore, the structural information strongly suggests that Rai1 and its mammalian
homolog Dom3Z may have enzymatic activity of its own.
Biochemical studies demonstrate that Rai1 possesses RNA 50-end pyrophospho-hydrolase activity, being able to remove a pyrophosphate group from RNA with
50-end triphosphate (pppRNA) (Xiang et al. 2009). Such an enzyme (RppH) was
first characterized in bacteria (Deana et al. 2008), which is a member of the Nudix
family of enzymes. Rai1/Dom3Z shares neither sequence nor structural homology
with RppH. Remarkably, Rat1 can stimulate this pyrophosphohydrolase activity
of Rai1, even though the binding site is located far from the active site of Rai1
(Fig. 7.7a) (Xiang et al. 2009).
Further biochemical studies showed that Rai1 can also remove unmethylated 50-end cap of RNAs (GpppRNA) (Jiao et al. 2010). This activity is however distinct
from the classical decapping enzymes. First, Rai1 has much lower activity toward
methylated 50-end cap. Second, the product released by Rai1 is GpppN, while the
classical decapping enzymes release m7GDP. Therefore, Rai1 appears to have two
distinct enzymatic activities.
The amino acid sequence of the Drosophila Rai1 homolog Cuff contains
mutations at several of the conserved acidic residues that are important for metal
ion binding. It is possible that Cuff does not have RNA 50-end pyrophospho-
hydrolase and decapping activities.
7 50-30 Exoribonucleases 183
The biochemical activities of Rai1 suggest a physiological function for this
enzyme. Rai1 may be an mRNA 50-end capping quality checkpoint. Both Rai1
substrates (pppRNA and GpppRNA) are intermediates in the mRNA 50-end cappingpathway. mRNAs with defective 50-end capping cannot serve as template for
translation. At the same time, these defective mRNAs cannot be degraded by
Xrn1/Rat1, due to the protected 50-end. Therefore, Rai1 can remove the 50-endfrom such mRNAs, and the products can then be rapidly degraded by Xrn1/Rat1.
Studies in yeast show that mRNAs with 50-end capping defects are stabilized in
cells lacking Rai1, consistent with this 50-end capping quality checkpoint model
(Jiao et al. 2010). In addition, mRNAs with aberrant 50-end capping also accumu-
late under stress conditions (glucose deprivation or amino acid starvation) in cells
lacking Rai1. Moreover, defective capping in yeast cells is linked to enhanced
recruitment of Rat1 throughout the transcribing unit, and promotes Pol II termina-
tion upstream of the poly(A) site (Jimeno-Gonzalez et al. 2010). This suggests that
Rai1 can remove the defective cap in such conditions, which then allows Rat1 to
Fig. 7.7 Rai1 possesses an active site of its own. (a) Schematic drawing of the structure of
S. pombe Rai1 (Xiang et al. 2009). A bound divalent cation in the active site is shown as a sphere.
The arrow points to residues in the interface with Rat1. (b) Molecular surface of the active site
region of Rai1, showing a large pocket. The metal ion is located at the bottom of the pocket.
(c) Overlays of the metal ion binding site in the structure of Rai1 (in black) and mouse Dom3Z
(in gray). Residue numbers in parenthesis are for Dom3Z. The interaction between Glu192 in
Dom3Z and the metal ion is mediated by a water molecule
184 J.H. Chang et al.
function as a torpedo to induce Pol II termination before the completion of
transcription, in a mechanism equivalent to that of transcription termination at the
30-end of the pre-mRNA.
These studies provide the first demonstration of an mRNA 50-end capping
checkpoint (Jiao et al. 2010; Xiang et al. 2009). It was generally believed in the
field that 50-end capping always proceeds to completion. The data on Rai1 convinc-
ingly demonstrate such a checkpoint in yeast. Dom3Z has a conserved active site,
and it remains to be seen whether such a checkpoint also functions in metazoans.
7.12 The 50-30 Exoribonuclease Rrp17
Rrp17 (ribosomal RNA processing) is associated with pre-ribosomes and the nuclear
pore complex (Oeffinger et al. 2009). It is an independent nuclease for the 50-endtrimming of the 5.8 S and 25 S rRNAs. Rrp17 is an essential gene in yeast, and has
highly conserved orthologs in most eukaryotes. Rrp17 has 50-30 exoribonucleaseactivity, with preference for a phosphate group at the 50-end of the substrate, while
a triphosphate group or cap structure inhibits the nuclease activity. In comparison to
the XRNs, Rrp17 also has activity toward RNAs with a 50-end hydroxyl group. The
activity requires Mg2+ ions, while the enzyme is inactive with Mn2+.
7.13 RNase J1/CPSF-73
Earlier studies have only identified 50-30 exoribonucleases in eukaryotes, leading tothe general belief that these enzymes are not present in prokaryotes. However, it
was recently discovered that the B. subtilis endoribonuclease RNase J1 also has
50-30 exoribonuclease activity, establishing for the first time the presence of such
activity in bacteria (Condon 2010; Mathy et al. 2007). The exoribonuclease activity
is required for mRNA degradation and for 50-end maturation of 16 S rRNA in
B. subtilis. Structural studies show that the endo- and exonuclease activities share
the same active site, and suggest that RNase J1 may switch from an endo mode to
exo mode on the same RNA substrate (de la Sierra-Gallay et al. 2008).
The exoribonuclease activity of RNase J1 is more permissive toward 50-endmodification of the RNA substrate as compared to Xrn1 (Mathy et al. 2007). The
highest activity is observed for RNAwith a 50-endmonophosphate or 50-end hydroxylgroup, although this activity is roughly tenfold lower than that of Xrn1, leading to the
suggestion that RNase J1 may require a cofactor for full activity. RNA with a 50-endtriphosphate group can also be degraded, but with roughly fourfold weaker activity.
The activity toward RNA with a 50-end cap is even lower (Mathy et al. 2007).
RNase J1 exists in a complex with RNase J2, which is a sequence homolog of
RNase J1 but with little nuclease activity. RNase J1 homologs are found in bacteria
(but not in E. coli), archaea (Clouet-d’Orval et al. 2010; de la Sierra-Gallay et al.
7 50-30 Exoribonucleases 185
2008), and they may also be present in the chloroplasts of plants (de la Sierra-
Gallay et al. 2008).
RNase J1 is a structural homolog of CPSF-73 (Mandel et al. 2006), the endoribo-
nuclease for the cleavage step in eukaryotic pre-mRNA 30-end processing (Mandel
et al. 2008; Proudfoot 2004; Wahle and Ruegsegger 1999; Zhao et al. 1999). Recent
studies suggest that CPSF-73 may also have an exoribonuclease activity, degrading
the downstream cleavage product of histone pre-mRNAs (Dominski and Marzluff
2007; Dominski et al. 2005; Yang et al. 2009). Analogous to the RNase J1/J2
heterodimer, the CPSF complex also contains CPSF-100, an inactive sequence
homolog of CPSF-73. It may be possible that mammalian CPSF-73/CPSF-100
and B. subtilis RNase J1/J2 share a common evolutionary origin.
7.14 Perspectives
Studies over the past few years have greatly enhanced our understanding of the
structure and function of 50-30 exoribonucleases, as well as identified new proteins
that possess this activity. It is anticipated that further characterization of these
enzymes in the coming years, especially in higher eukaryotes, will lead to signifi-
cant new insights into the biological significance of these enzymes. Moreover,
studies on Rat1–Rai1 complex led to the discovery of a novel mRNA 50-endcapping quality checkpoint. There may be further exciting surprises from the
studies of these exoribonucleases and their interaction partners.
Acknowledgment This research is supported in part by grants from the NIH to LT (GM077175).
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